Capture, disruption, and extraction apparatus and method

ABSTRACT

A cell capture, disruption, and extraction method includes a introducing a plurality of abrasives in a disruption chamber, which can include diamond powder, variably and multi dimensionally disbursed therein, and a pestle positioned in the disruption chamber. The method includes agitating the abrasives by moving the disruption chamber and/or pestle, agitation of the abrasives tearing cell structure in the solution to access its contents. A binding column or size exclusion column can be positioned downstream of the disruption chamber. Cell solution can first be introduced in the disruption chamber, the abrasives capturing the cells and allowing therethrough and purging the waste content, then breaking the cell content. The lysate can then bind to an extraction matrix downstream of the disruption chamber or it can be mixed in with the abrasives.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

A portion of the disclosure herein was made with Government supportunder Contract No. NNA09DA96C by The National Aeronautics and SpaceAdministration. The government has certain rights in the disclosure.

BACKGROUND Technical Field

The present disclosure generally relates to cell capture, disruption,and extraction, and more particularly, to a device and method forreceiving, isolating, and preparing cell lysate and extraction ofspecific cell content such as nucleic acid, proteins, lipid and thelike.

Description of the Related Art

Typical starting materials for genomics or proteomics assays arebiological samples. These samples could include, among others, plant oranimal tissue, cultured bacteria, yeast, algae, blood and/or otherbodily fluids. To extract any part of the cellular components from thesesamples, the cells are typically removed from media in which they exist,such as growth or storage solution, or otherwise concentrated into asmall volume. The cell membrane and/or cell walls are then broken toexpose its content (lysis). Some cells may have a thin membrane that canbe broken by simply exposing them to chaotropic salts such asguanidinium thiocyanate. Other cells, such as plant and single cell bluegreen algae, have thicker cell membranes or additional cell walls,making them difficult to break. Bacteria spores such as bacillus,subtilis or anthracis form strong bacteria spores, which are alsodifficult to break.

Furthermore, typically a targeted material, which can be DNA, RNA,Protein, lipids, or other cellular material, is extracted throughmethods such as affinity columns from prepared cell lysate.

Existing methods to perform the steps above are carried out usingdistinct individual tools and kits performing these steps separately,and their technologies for performing these steps do not lend toperforming combination-processing steps. For example, filter membranesare used to separate the cells from its surrounding media. Harshchemical, heat, Sonication, Freeze/Thaw and/or bead beating are used tobreak cells open.

Commercially available filters currently used for cell separation orconcentration purposes, are slow and eventually clog. Furthermore,existing devices typically collect cells from the filter, then remove itand move it to a separate lysis tube. This transport can be prone tohandling errors and contamination, is labor intensive, and reducesoverall lysis yield and quality.

Therefore, existing devices consume excess time and expense to performcomplete sample preparation and target material extraction, and areprone to contamination and/or destruction of intracellular componentsbeing sought. Furthermore, existing methods may not be reproducible; maybe applicable to only one of small scale or large scale processing; canexhibit high noise levels, yield variability, and generation of freeradicals; can only be applicable to easily breakable cells; may not workwith microorganisms; are slow and susceptible to clogging; and/orrequire expensive, complicated, super high speed breakup mechanism,and/or be prone to contaminating external elements.

BRIEF SUMMARY

According to one embodiment, a method for processing cells from cellsolution having cells and waste matter includes placing a plurality ofabrasive particles in a disruption chamber, at least partially placing apestle in the disruption chamber, introducing cell solution includingcells and waste matter into the disruption chamber following placing theabrasives therein, selecting the abrasives to embody sizes and shapes toobstruct the cells in a space between the abrasives, and allowingtherethrough the waste matter following introduction of the cellsolution to the disruption chamber, and agitating the cells against theabrasives by actuating at least one of the disruption chamber and thepestle, the selected sizes and shapes of the abrasives configured to cutthe cells separated from the waste matter.

According to one aspect, the method includes arranging the abrasiveparticles in at least a first layer having a first density of abrasiveparticles and a second layer having a second density of abrasiveparticles, the first density being different from the second density.

According to one aspect the method includes communicating the cut cellsto a binding matrix.

According to one aspect, the placing the plurality of abrasives includessubstantially filling the disruption chamber with the abrasives.

According to one aspect, the method includes positioning a bindingmatrix between an outlet of the disruption and an outlet port in fluidcommunication with the disruption chamber.

According to one aspect, the method includes substantially retaining theabrasives between a first mesh component toward a first end of thedisruption chamber and a second mesh component toward a second end ofthe disruption chamber.

According to another embodiment, a method of manufacturing an apparatusfor cell capture, disruption, and extraction from a cell solution havingcells and waste matter, includes forming a disruption chamber in a firstfitting, at least one of (i) forming an outlet port in the first fittingand (ii) coupling a second fitting to the first fitting and forming theoutlet port in the second fitting. The method according to an aspectfurther includes the outlet port being configured to be in fluidcommunication with the disruption chamber, forming an inlet port influid communication with the disruption chamber, and providing aplurality of abrasives configured for placement in the disruptionchamber prior to introduction of the cell solution to the disruptionchamber, sizing and shaping at least some of the abrasives to allowtherethrough the waste matter and to obstruct the cells in a spacebetween the abrasives the cells, providing a pestle configured to be atleast partially received in the disruption chamber, providing anactuation device configured to operatively couple to at least one of thedisruption chamber and the pestle to impart motion thereto, such motionagitating the abrasives, at least some of the abrasives being comprisedof a material and shape to cut the cells separated from waste matter, inresponse to the agitation.

According to one aspect, the providing the plurality of abrasivesincludes arranging the plurality of abrasives in a plurality of layersforming a multi-dimensional filtration and grinding matrix, theplurality of layers having at least one of distinct abrasive sizes, andshapes, at least one of the layers having the abrasives allowingtherethrough the waste matter and obstructing the cells in a spacebetween the abrasives, and at least one of the layers having theabrasives comprised of a material and shape to cut the cells.

According to one aspect, the method includes positioning at least oneretaining member adjacent the disruption chamber.

According to yet another embodiment, a method for extracting cell matterfrom a cell suspension having cell matter and non-cell matter includesat least partially positioning a pestle in a cavity, loading a pluralityof abrasives in the cavity having an inlet and an outlet, introducingthe cell suspension to the cavity following the loading of the pluralityof abrasives, selecting the abrasives respectively having sizes andshapes such that at least some of the abrasives obstruct the cell matterin a space between the abrasives, filtering the cell matter from thecell suspension and allowing therethrough non-cell matter followingintroduction of the cell suspension to the cavity, and imparting motionto at least one of the pestle and the cavity to agitate the abrasives,the sizes and shapes of at least some of the abrasives configured to cutthe cells separated from the waste matter.

According to one aspect, the loading the plurality of abrasives in thecavity includes arranging the plurality of abrasives in at least a firstlayer and a second layer, selecting the abrasives of the first layer tobe larger than the abrasives in the second layer.

According to one aspect, the loading the plurality of abrasives in thecavity includes arranging the plurality of abrasives in a plurality oflayers including first, second, and third layers, selecting theabrasives of the second layer to be smaller than the abrasives of thefirst and third layer, and arranging the second layer between the firstand third layers when the plurality of abrasives are loaded in thecavity.

FIGURE BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a side schematic view of a capture, disruption, and extractionapparatus according to one embodiment.

FIGS. 2A through 2D are each a cross-sectional view of the apparatus ofFIG. 1, taken across section 2A, 2B, 2C, 2D, according to variousembodiments.

FIG. 3A is a side schematic view of a portion of the apparatus of FIG. 1according to one embodiment.

FIG. 3B is a side schematic view of a portion of the apparatus of FIG. 1according to another embodiment.

FIG. 4 is a side schematic view of a portion of the apparatus of FIG. 1according to another embodiment.

FIG. 5 is a side view of a capture, disruption, and extraction apparatusaccording to one embodiment.

FIG. 6 is an exploded isometric view of the apparatus of FIG. 5according to one embodiment.

FIG. 7 is a cross-sectional view of the apparatus of FIG. 6, takenacross section 7, according to one embodiment.

FIG. 8 is a portion of a capture, disruption, and extraction apparatusaccording to one embodiment.

FIG. 9 is a side view of a capture, disruption, and extraction apparatusaccording to one embodiment.

FIGS. 10A and 10B are respectively side and bottom views of a pestle ofthe apparatus of FIG. 9, according to one embodiment.

FIGS. 11A and 11B are respectively side and bottom views of a pestle ofthe apparatus of FIG. 9, according to another embodiment.

FIG. 12 is a capture, disruption, and extraction apparatus according toone embodiment.

FIG. 13 is a side exploded view of a capture, disruption, and extractionapparatus according to one embodiment.

FIG. 14 is a side view of a portion of the capture, disruption, andextraction apparatus of FIG. 13 according to one aspect.

DETAILED DESCRIPTION

In one embodiment as illustrated in FIG. 1, a capture, disruption andextraction apparatus 100 includes a solution inlet port 103 and a celldisruption chamber 104 in fluid communication with the inlet port 103.In the illustrated embodiment of FIG. 1, the inlet port 103 is in directfluid communication with the cell disruption chamber 104; however, inother embodiments, the inlet port 103 can be indirectly in fluidcommunication with the cell disruption chamber 104. In one aspect,instead of, or in addition to the inlet port 103, the apparatus 100 caninclude an auxiliary inlet/outlet port 107 formed a portion of theapparatus 100, which in turn is coupled directly or indirectly to thecell disruption chamber 104.

For example, according to one aspect, the auxiliary port 107 can be influid communication with the outlet port 105 where cell solution orabrasive content similar to that described above can first be introducedin the disruption chamber 104 prior to operation of the apparatus 100 tofilter cell solution and sequentially exit the waste and filteredcontent out through outlet port 105. In some embodiments, the apparatuscan be unassembled to introduce abrasives as later described.

In some aspects, the auxiliary port 107 can communicate waste orfiltrate out of the apparatus 100 or away from the chamber 104 andoutlet port 105, and/or allow abrasives and/or solution toward thechamber 104. Other embodiments may use the auxiliary port 107 tointroduce wash fluid to a downstream location as will be described withrespect to a binding matrix below.

In one aspect, the apparatus 100 can include an actuation device 108,which in some embodiments can be manual and/or automatic. For example,in some aspects, the actuation device 108 can include a motor. Forclarity of description and without any intention to limit the scope ofthe present disclosure or the actuation device, the actuation device 108will hereinafter be referred to as the motor 108.

The motor 108 can be operatively coupled with respect to the celldisruption chamber 104, for example via a shaft and/or a pestle, and/ora combination thereof, or any other suitable coupling member, to rotate,oscillate, move, or otherwise shake or agitate contents in thedisruption chamber 104. In one aspect, the motor 108 can include or becoupled to a pestle 110 at least partially extending in the disruptionchamber 104 as illustrated in FIGS. 1 and 2A through 2D, and configuredto be rotated or moved therein, or effect rotation or movement of thedisruption chamber 104. In one embodiment, for example, the motor 108can be operatively coupled to the pestle 110 via an intermediate member106 that can include or be a shaft, gear mechanism, screw or ball screwmechanism, any combination thereof, or any other suitable drive orrotation mechanism, system, or structure.

In one embodiment, a portion of the pestle 110 adapted to be received inthe disruption chamber 104 can include a key member, a geometric shape,and/or a frictional feature or attribute, complementary to that formedin, coupled to, or attached to or on an outer surface thereof. In someembodiments, the pestle 110 can include an auger.

According to one aspect, the disruption chamber 104 includes a cavity112 configured to receive the pestle 110, and a volume 114 between thepestle 110 and an outer wall 116 of the disruption chamber 104. In anaspect the volume 114 is adapted to receive fluid, liquid, solids,particles and/or any combination thereof, for example, a cell solutionand/or abrasives as will be discussed with respect to certainembodiments below. In various embodiments, the pestle 110 can include adifferent shape and/or periphery to promote disruption and/or agitationin the volume 114.

For example, in the illustrated embodiment of FIG. 2A, a pestle 110A hasa hexagonal outer surface configured to be received in a cavity 112A toform a volume 114A about the periphery of the outer surface of thepestle 110A. In other embodiments, the pestle 110 may include adifferent shape. For example, as illustrated in FIGS. 2B through 2D,pestles 110B, 110C, 110D can include an elliptical, cross, or starshaped outer periphery, respectively, forming volumes 112B, 112C, 112D,respectively about the periphery of the respective pestles 110B, 110C,110D.

Various embodiments may include various forms of actuation. For example,the motor shaft can be coupled to an end of the disruption chamberand/or the pestle in some embodiments. In yet other embodiments, therotating portion of the motor can surround or circumscribe thedisruption chamber and/or the pestle to rotate one with respect to theother, or two rotate them in opposite directions. These and othersuitable actuation mechanisms are contemplated to be within the scope ofthe present disclosure. In some embodiments, the motor can be coupled tothe disruption chamber to impart motion thereto and the pestle can bestationary while the disruption chamber rotates when actuated. In yetsome embodiments, the pestle and the disruption chamber can be actuatedto rotate in substantially opposite radial directions.

FIG. 3A illustrates the volume 114 of an apparatus 100 according to oneembodiment with the pestle 110 not shown for clarity of illustration. Inone embodiment, the apparatus 100 includes an abrasive content 118 inthe volume 114. In one aspect, in an application of the apparatus 100,cell solution is introduced via the inlet port 103, and fluidlycommunicated to the disruption chamber 104. In some embodiments, thedisruption chamber 104 can be prefilled with the abrasive content 118.In an aspect, as the cell solution enters the disruption chamber 104,and negotiates its way through the abrasives 118, the abrasives 118serve to guide and filter the solution, allowing through non-cellmatter/fluid and isolating cells by trapping or obstructing them in thespace formed between the abrasive particles.

In various embodiments, the abrasives 118 can be layered inmultidimensional configuration, for example, a three dimensionalconfiguration to facilitate guiding the cell solution in a first phase,and filtering it in a second phase or third phase. For example, theabrasives 118 can include a plurality of abrasive type, size or kindand/or a plurality of abrasive density and/or quantity. For example, inone embodiment, the volume 114 in cavity 112 may receive or house agroup or layer of abrasives of a larger size 115, facilitating guidingthe solution, adjacent a group or layer of abrasives of a medium size117, which in turn is adjacent a group or layer of abrasives of asmaller size 119, which facilitates filtration/isolation of the cellfrom the cell solution. For better context, further below an example isprovided as one embodiment for a thorough understanding of an embodimentof the present disclosure without any intent to limit the scope of thedisclosure.

Some embodiments may include structure, components, and/or features topromote containment of the abrasives 118 in the chamber 104. Forexample, the apparatus 100 may include mesh and/or perforated layers orcomponents 123, 125 positioned on opposing sides of the abrasives 118.For example, the mesh components 123, 125 can include a PEEK® mesh orother suitable fine mesh having a plurality of orifices or openings tocontain the abrasives, such as 35 micron openings in some aspects.

In the description that follows and generally throughout thisdisclosure, nonlimiting examples of operation are described with respectto solutions being processed through various embodiments herein.Generally, separation of undesired solution content and desired solutioncontent will be described. It is understood by those of skill in the artthat the specifics of such content can vary. For example, the undesiredcontent can include water, growth media, storage solution, waste matter,or any other content the user may desire to separate to isolate thedesired content. The desired content can include cell content such asnucleic acid, proteins, lipid and the like. For clarity of description,the desired content will be referred to as cells or cell content and theundesired content will be referred to as waste matter, without anyintention to limit the disclosure to any specific desired or undesiredcontent.

In an aspect, solution such as cell solution is introduced to thechamber 104 through inlet port 103. In one aspect, the abrasives 118obstruct, capture, and/or bind to desired fluid portions from the cellsolution and substantially isolate the cells, while waste matter ispurged through the outlet port 105 and/or the auxiliary port 107. Inembodiment, when the waste matter is purged, the motor 108 actuate thepestle 110, initiating agitation and breaking up the cells fromagitation through contact with and between the pestle 110, abrasives118, and/or walls of the disruption chamber 104. In one embodiment, theabrasives 118 further serve to promote breaking open the cells as themotor 108 moves, rotates, oscillates, and/or otherwise agitates orshakes the pestle 110 and/or the cell disruption chamber 104, forcingthe abrasives to shear, rub, or scrape against the cells, breaking themup and forming a desired broken cell slurry. Such cell slurry or lysatecan then be fluidly communicated through the outlet port 105 via a washstep where fluid communicates the lysate away from chamber 104 toward adesired downstream destination.

In some embodiments, the abrasives can be selected from a group ofmaterials that embody sharp edges, facilitating tearing cells even atslower speeds, thereby requiring less power, exhibiting less noise, moreeffectively breaking cells, even in micro applications, and operating atlesser speeds as compared to existing devices using purely shearingand/or bombarding or beating techniques. In some embodiments, theabrasives include diamond particle or diamond dust. In some embodiments,the abrasives can include, glass shreds, aluminum oxide, silicon dioxideor other suitable powder of appropriate size.

In some embodiments, the abrasive layers may be repeated in the sameorder to increase efficacy of the foregoing process. In someembodiments, the abrasive layers could include other combination of thelayers. For example, the layers can progress from toward a first end ofthe chamber 104 to toward a second end of the chamber 104, opposing thefirst end, the abrasive layers can range sequentially in size and fromlarge, to medium, to small, to medium, to large.

In some embodiments, the density or quantity of the abrasive orabrasives toward opposing terminal ends of the disruption chamber 104can be greater than that toward the center of the chamber 104. In someembodiments, the variation in abrasive density and/or quantity from theopposing terminal ends of the disruption chamber 104 toward the centerthereof can vary, and in one aspect, increase or decrease. For example,the abrasive content in the chamber 104 may include three abrasivelayers from the terminal ends toward the center of the chamber 104. Insuch an aspect, the outer most layers closest to the terminal ends ofthe disruption chamber 104 can include abrasive of larger sizedistributed at a coarser spacing as compared to the same in the innertwo layers toward the disruption chamber 104 central region.

For example, in one embodiment, as illustrated in FIG. 3B, the abrasives118 in the chamber cavity 112 of chamber 104 can include outer layers ofthe abrasives of the larger size 115, intermediate layers of abrasivesof the medium size 117 and a central layer of abrasives of the smallsize 119.

For better context, further below an example is provided as oneembodiment for a thorough understanding of an embodiment of the presentdisclosure without any intent to limit the scope of the disclosure.

Therefore, embodiments of the present disclosure facilitate receiving,isolating, and breaking cells while mitigating the number and complexityof components used, and in an integrated process, to isolate cells andbreak them open exposing their content.

In some embodiments, as illustrated in FIG. 1, the apparatus 100 mayinclude a solid phase extraction matrix, size exclusion matrix, or othersuitable target specific capture matrix 120 configured to furtherisolate particular matter or material from the broken cells, such asRNA, DNA, protein, lipids or any combination thereof, and/or any othersuitable matter or material desired to be extracted. In someembodiments, the matrix 120 can include a composite or other filteringmaterial such as fiberglass, glass, sand, any combination thereof,and/or any other suitable frit, matrix, and/or binding or filteringmaterial. In one aspect, the matrix 120 can include a Qiagen® RNAextraction membrane.

In some aspects, the extraction matrix 120 can be in fluid communicationwith the cell disruption chamber 104, for example with an outlet 105thereof, the matrix 120 receiving the lysate following the aboveabrasive cell breaking process, further processing the lysate to extracta desired target, such as RNA, DNA, protein, lipids, any combinationthereof, and/or any other suitable matter or material desired to beextracted. The auxiliary port 107 in one embodiment can also allow washsolution to be introduced to matrix 120, for example following thelysate having been communicated to the matrix 120.

In an aspect, as illustrated in FIG. 1, the apparatus 100 may furtherinclude a target outlet port or orifice 122 in fluid communication withthe matrix 120 and adapted to receive the target matter or material andcommunicate it to an environment external to the apparatus 100 for usein a column, or in experiments and/or application in a subsequentchemical, biochemical, biotechnical, genomic and/or proteomic context.

In addition, or instead, as illustrated in FIG. 4, in another aspect,the apparatus 100 may include an extraction matrix 121 between theoutlet port 105 and the pestle 110.

FIGS. 5, 6, and 7 illustrate an embodiment of a capture, disruption, andextraction apparatus 200 with some internal components shown in hiddenlines. Not all internal components may have been shown in all theseFigures for clarity of illustration. According to one embodiment, theapparatus 200 includes a solution inlet port 203, a cell disruptionchamber 204 in fluid communication with the inlet port 203, and anoutlet port 205 in fluid communication with the disruption chamber 204.

In one embodiment, the inlet port 203 is in direct fluid communicationwith the cell disruption chamber 204; however, in other embodiments, theinlet port 203 can be indirectly in fluid communication with the celldisruption chamber 204.

In one aspect, instead of, or in addition to the inlet port 203, theapparatus 200 can include an auxiliary inlet/outlet port 207 formed aportion of the apparatus 200, which in turn is coupled directly orindirectly to the cell disruption chamber 204.

For example, according to one aspect, the auxiliary port 207 can be influid communication with the outlet port 205 where cell solution orabrasive content similar to that described above can first be introducedin the disruption chamber 204 prior to operation of the apparatus 200 tofilter cell solution and sequentially exit the waste and cells outthrough outlet port 205. In some aspects, the auxiliary port 207 cancommunicate waste or filtrate out of the apparatus 200 or away from thechamber 204 and outlet port 205, and/or allow abrasives and/or solutiontoward the chamber 204, or allow washing solutions to be introduce tomatrix 220, for example following lysate and/or broken cell slurryhaving been communicated to the disruption chamber matrix 220 fromchamber 104.

In one aspect, the apparatus 200 can include an actuation device 208,which in some embodiments can be manual and/or automatic. For example,in some aspects, the actuation device 208 can include a motor. Forclarity of description and without any intention to limit the scope ofthe present disclosure or the actuation device, the actuation device 208will hereinafter be referred to as the motor 208.

The motor 208 can be operatively coupled to at least one of the celldisruption chamber 204 or the pestle 210, for example via a shaft or anyother suitable coupling member, to rotate, oscillate, move, or otherwiseshake or agitate the disruption chamber 204. In one aspect, the motor208 can include or be coupled to the pestle 210, which at leastpartially extends in the disruption chamber 204, and configured to berotated or moved therewith, or effect rotation or movement of the pestle210.

For example, the motor 208 can be coupled to the pestle 210 via acoupling member 209, which in one aspect can include a coupling shaft211. In one aspect, the coupling member 209 can include a securingdevice 213 removably securing the shaft 211. For example, the securingdevice 213 can include a security screw, pin, or the like.

In one embodiment, a portion of the pestle 210 is adapted to be receivedin the disruption chamber 204. According to one aspect, the disruptionchamber 204 includes a cavity 212 configured to receive the pestle 210,and a volume 214 between the pestle 210 and an outer wall 216 of thedisruption chamber 204. In an aspect the volume 214 is adapted toreceive abrasive layers and/or cell solution, for example, in oneaspect, abrasive layers, followed by cell solution. In variousembodiments, the pestle 210 can include a different shape and/orperiphery to promote disruption and/or agitation of contents in thevolume 214 as discussed above.

In one embodiment, in operation, cell solution can be introduced in thedisruption chamber 204 via the inlet port 203 and/or the auxiliary inletport 207, and disrupted through relative motion of the pestle 210 withrespect to the disruption chamber 204.

In an aspect, abrasives can be introduced as discussed above in moredetail to capture, obstruct, or trap cells and purge unwanted fluidportions or waste from the cell solution and substantially isolate thecells. In an aspect during or following purging of such waste matter,when the motor 208 actuates the pestle 210 and/or the disruption chamber204, it induces breaking up the cells from agitation through contactwith and between the pestle 210, abrasives 218, and/or walls of thedisruption chamber In one embodiment, the abrasives further serve topromote breaking open the cells as the motor 208 moves, rotates,oscillates, and/or otherwise agitates or shakes the pestle 210 and/orthe cell disruption chamber 204, forcing the abrasives to shear, rub, orscrape against the cells, breaking them up and forming a desired brokencell slurry or lysate. Thereafter such slurry or lysate can becommunicated and/or washed through the outlet port 105 to a desireddestination for various desired applications.

In some embodiments, as illustrated in FIGS. 6 and 7, the apparatus 200may include a solid phase extraction matrix, size exclusion matrix, orsimilar matrix 220 configured to further isolate particular matter ormaterial from the broken cells, such as RNA, DNA, protein, lipids or anycombination thereof, and/or any other suitable matter or materialdesired to be extracted. In some embodiments, the matrix 220 can includea composite or other filtering material such as fiberglass, glass, sand,any combination thereof, and/or any other suitable frit, matrix, and/orbinding or filtering material. In one aspect, the matrix 220 can includea Qiagen® RNA extraction membrane.

In some aspects, the extraction matrix 220 can be in fluid communicationwith the cell disruption chamber 204, for example with an outlet 205thereof, the matrix 220 receiving the lysate following the aboveabrasive cell breaking process, further processing the lysate to extracta desired target, such as RNA, DNA, protein, lipids, any combinationthereof, and/or any other suitable matter or material desired to beextracted. In some embodiments, the material of which the matrix 220 isconstructed such as silicon dioxide can be added in powder form and usedin the chamber 204 in addition to, or instead of, the abrasives or bethe abrasive.

In an aspect, the apparatus 200 may further include a target outlet portor orifice 222 in fluid communication with the matrix 220 and adapted toreceive the target matter or material and communicate it to anenvironment external to the apparatus 200 for use in experiments and/orapplication in a subsequent chemical, biochemical, biotechnical, genomicand/or proteomic context.

In various embodiments, suitable structures and components can be usedto house and/or form the foregoing features. For example, in oneembodiment as illustrated in FIG. 6, the apparatus 200 includes adisruption fitting or housing 232 forming or containing the disruptionchamber 204 and the inlet port 203; an outlet fitting 242 forming and/orhousing the outlet port 205 and the auxiliary inlet/outlet 207; aclamping fitting 230; and an egress fitting 246. In some embodiments,the clamping fitting 230 and/or the egress fitting 246 where included,can form the target outlet port or orifice 222. In one embodiment, thehousing 232, the motor 208, the outlet fitting 242, clamping fitting230, and egress fitting 246, can be removably coupled to allowdisassembling the apparatus 200.

As illustrated in FIG. 6, some embodiments may include a containmentassembly 224 to contain the abrasives in the disruption chamber 204. Inone aspect, the containment and filtering assembly 224 can include amesh component 226 such as a PEEK® mesh or other suitable fine meshhaving a plurality of orifices or openings to contain the abrasives.

In some embodiments, the containment assembly 224 may further include afilter positioned adjacent the mesh component 226, further finefiltering the disruption chamber contents. In some aspects, theapparatus 200 may include a seal or O-ring 228 adjacent the containmentand filtering assembly 224, securing that assembly. In some embodiments,the apparatus 200 may include a seal or O-ring 229 between the pestle210 and disruption chamber 204. In some embodiment the apparatus 200includes a mesh element below the first layer of abrasives.

In some embodiments, the apparatus 200 includes the housing 232 in whichthe disruption chamber 204 is formed. In some aspects, the housing 232further includes a gutter, depression, recess, or the like, configuredto receive the containment and filtering assembly 224. In some aspects,the apparatus 200 can include the clamping fitting 230 configured to becoupled, fixedly or removably, to the housing 232, and having formedtherein the outlet port 222, which can include a cavity, recess,channel, cylindrical opening, and/or any combination thereof, or othersuitable port structure. In some aspects, the clamping fitting 230 mayinclude grooves or channels 234 configured to promote or facilitatedraining of fluid or cell slurry exiting the disruption chamber 204.

In some embodiments, the housing 232 may include a cavity, recess,channel, cylindrical opening, and/or any combination thereof, or othersuitable port structure, forming the auxiliary inlet/outlet port 207 forintroducing solution for being processed as disclosed herein.Alternative embodiments may include a distinct assembly or componentcoupled to the housing 232 to fluidly communicate therewith andintroduce solution to the inlet 203 of the disruption chamber 204.

In one embodiment, the outlet fitting 242 and clamping fitting 230 canbe removed to allow loading the chamber 204 with abrasives, then placingthe containment assembly 224 to cover the chamber 204 and securingoutlet fitting 242 and clamping fitting 230 to the disruption fitting232 to prepare for introducing cell solution through inlet port 203.

In various embodiments, these features may be formed on either of thefittings or in some embodiments the fittings can be integrated andformed from a unitary body of material.

Various embodiments may include any suitable stabilizing, mounting,housing, and/or fluid feeding components for the apparatus 200. Forexample, the apparatus 200 can include an outer housing including firstand second valve manifolds for communicating fluid in to and out of thedisruption chamber 204.

Embodiments of the present disclosure therefore facilitate filtering andretaining cells and extracting their content in a simultaneous and/orsequential manner, thereby increasing efficiency and processing speedswhile decreasing costs, labor, and chance of contamination. In addition,embodiments of the present disclosure can be used for additionalprocessing such as washing samples with buffer solution and/or isolatingactive agent introducing contents exiting the disruption chamber to abinding matrix or frit.

The following example is provided for a thorough understanding of anembodiment having features according to some aspects, and makesreference to certain portions of the various embodiments and aspectsdiscussed above; however, it is understood the present disclosure is notlimited to what is described for this example and other embodiments arecontemplated.

Other applications and combination of embodiments herein, for example,various abrasives, material, shapes, and actuation mechanisms, amongother features, are contemplated to be within the scope of the presentdisclosure.

For example, in some embodiments, a cell capture, disruption, andextraction apparatus may include as its abrasive particles, silica,silicon dioxide, glass fiber or material similar or substantiallyidentical to the solid phase extraction membrane/matrix, instead of, orin addition to, diamonds. In such an embodiment, a separate solid phaseextraction matrix can be omitted, these particles performing disruptionand extraction in the disruption chamber.

Furthermore, an apparatus according to various embodiments does not haveto be limited to the specific combinations and features specificallydescribed herein, and can be scaled or modifying without deviating fromthe scope of the present disclosure, to be suited for particularapplications.

For example, in an embodiment illustrated in FIGS. 8 and 9, a celldisruption device 300 can include a column 301 having a disruptionchamber 304 substantially filled in a similar or the same manner asdiscussed with respect to other embodiments herein, with a spectrum ofabrasive particle layers, for example in one aspect, from largeparticles 315 to medium particles 317 to small particles 319, from andbetween opposing portions and/or mesh elements 303, 305, of a portion ofdisruption chamber 304 toward a middle, intermediate, lower, or centralregion 307 thereof. The disruption chamber 304 can be fabricated from atube, column, or any other suitable shape or material.

In one application, the cell sample can be introduced into thedisruption chamber 304 according to any of the methods described hereinor using a pipette to introduce the sample to a standalone disruptionchamber 304, or in a manner as disclosed in U.S. patent application Ser.No. 14/335,946. In one aspect the cells can be induced into thedisruption chamber 304 using any suitable biasing mechanism. Forexample, in one embodiment, the apparatus 300 may include a vacuumdevice to apply a vacuum to the bottom or toward an end or side of thedisruption chamber (negative pressure), or a blowing device such as asyringe or pump, to apply positive pressure from an end or side of thedisruption chamber, to induce the sample through the abrasive particles.

In addition, or instead, of the foregoing biasing devices, in someaspects, the apparatus 300 can include a centrifuge device to inducemovement of the sample through the abrasives, and trap cells between theabrasive. In one embodiment as illustrated in FIG. 9, the apparatus 300includes a pestle 310 including a plurality of agitation members 311.The agitation members 311 can include any suitable disrupting shape orfeature, such as conical extrusions such as thorn-like extrusions, atits end. In one aspect, the pestle 310 can be manually and/orautomatically actuated to press it into the abrasive layers 315, 317,319. The apparatus 300 can in one aspect be configured or include amanual and/or automatic actuation device to rotate the pestle 310 and/orthe disruption chamber 304 to move or turn the abrasives and cell samplewith respect to the agitation members 311, thereby disrupting orbreaking the cells to reach its content.

Such an embodiment can combine features of other embodiments, such asrouting the cell content through a solid phase extraction matrix toreach a target active agent, and/or including a containment andfiltering assembly similar to that described above including a mesh toretain abrasives in the disruption chamber 304.

In some embodiments, the apparatus 300 may include a solid phaseextraction matrix 320 positioned downstream the abrasive content 318 andconfigured to further isolate particular matter or material from thebroken cells, such as RNA, DNA, protein, lipids or any combinationthereof, and/or any other suitable matter or material desired to beextracted. In some embodiments, the matrix 320 can include a compositeor other filtering material such as fiberglass, glass, sand, anycombination thereof, and/or any other suitable frit, matrix, and/orbinding or filtering material. In one aspect, the matrix 320 can includea Qiagen® RNA extraction membrane.

Other pestle arrangements can be incorporated in various embodiments andcontemplated to be within the scope of the present disclosure. Forexample, FIGS. 10A and 10B illustrate a pestle 313 having a hexagonalagitation member 315. Other configurations are possible. For example,FIGS. 11A and 11B illustrate another embodiment including a pestle 317having an elliptical agitation member 319. Other features can beincorporated in various aspects to bring about additional agitationand/or disruption of cell solution. For example, the agitation member319 can include ridges or other suitable agitation features 321 on aperiphery thereof. In yet another aspect as illustrated in FIGS. 12, 13,and 14, the solid phase extraction matrix 320 can be in an outer column302 in which the column 301 is configured to nest. For example, thecolumn 301 can be in fluid communication with the outer column 302 tocommunicate thereto, the filtered cell slurry following agitation in theabrasive content 318, to then be further filtered through the matrix 320positioned downstream in the outer column 302. In one embodiment, thepestle 310 can be operated to disrupt and/or agitate contents of thecolumn 301 in a first step as illustrated in FIG. 12, then the column301 containing the cell slurry being transferred to couple with theouter column 302 to communicate the slurry thereto and toward the matrix320 in a second step.

Example

In on example, abrasive particles form a three-dimensional filter in thevolume surrounding the pestle inside the disruption chamber, whichchamber can be formed from a cylindrical cavity. A fine mesh (e.g., PEEKmesh) is positioned at either end of the disruption chamber to preventor substantially mitigate the particles from escaping the capture anddisruption chamber. For improved cell capture and grinding and/ordisruption setup, the setup can in one example be mounted substantiallyvertically promoting the particles settling into place when loaded. Adonut-shaped mesh is positioned on or toward the lower end of thechamber such that the inner hole of the donut fits snugly around themotor shaft, which actuates or rotates the pestle.

A containing and filtering assembly including a screen is positionedadjacent the lower end of the disruption chamber. With the bottom screenin place, which can also be positioned around an axle, the disruptionchamber being screwed into, otherwise coupled to, or having an innerhollow portion surrounding the axle. A funnel is placed on top of thechamber to facilitate abrasive particle loading and then the chamber isfilled with water using a syringe that is attached to the fluidic inletof the disruption chamber. After clearing any bubbles in the chamber,the particles settle. Finally, the abrasive particles, suspended inwater or other fluid, are loaded into the chamber with a pipette. Thelargest particles are loaded first and last, and the smallest particlesare loaded as the middle layer. The particles are loaded in discretevolumes (e.g., 30 μl at a time) to enable substantially completesettling and packing. For example, in one embodiment for isolating andgrinding cyanobacteria, diamond dust with average particles sizes of 60μm, 30 μm, and 15 μm is loaded 30 μl at a time. Approximately, 60 μl of60 μm diamond dust forms the bottom layer, followed by 30 μl of 30 μmdiamond dust, 30 μl of 15 μm diamond dust, 30 μl of 30 μm diamond dust,and enough 60 μm diamond dust to fill the rest of the chamber (˜60-120μl).

Sufficient time is allowed between each 30 μl loading for the particlesto substantially settle in the chamber. After the initial loading, thesyringe at the fluidic inlet of the chamber can be pulled back gentlyand slowly to help pack the diamond dust, and mitigate or preventdisrupting the layers. The chamber is topped off with 60 μm diamond dustto ensure the chamber is substantially completely full. It isadvantageous to substantially fill the chamber because if there isexcess space, channels or burrows may form in the diamond dust due tofluid flow; such channels can disrupt the filtration process as themajority of fluid, including cell suspensions, flows through the channelwithout being filtered.

Once the chamber is substantially full with abrasive particles, theremainder of the water left in the chamber should be pulled out usingthe syringe. The funnel is removed, a PEEK mesh is placed on top of thechamber to substantially mitigate or prevent particles from spilling outthrough the outlet port, and the disruption chamber cap is mounted.

Prior to cell loading, the disruption chamber can be washed and primed,for example, with a cell lysis and nuclease-deactivating reagent. Afterthe chamber is washed and primed (if needed), the cell suspension isflowed through the disruption chamber. Flow through the chamber prior togrinding can be maintained at low rates (e.g., 2 μl/min) tosubstantially not disturb the layers of abrasive particles forming thethree dimensional filter. As the cells are captured in the filter formedby abrasives, the media continues through the device to the outlet whereit can be sent to waste. Being three dimensional, the filter is capableof capturing a large number of bacteria as compared to two-dimensionalfilters. The captured cells can then be exposed to various reagentsflowed through the chamber at low rates. In the example of RNAextraction from cyanobacteria, the cells can be exposed to a cell lysisbuffer diluted with ethanol. The lysis buffer helps to substantiallydisrupt the durable cell walls of the bacteria, and the ethanol isincluded since it promotes the attachment of RNA to the silica gel RNAcapture matrix.

Once the cells are ready for grinding, the motor is turned on whichactivates the stirring mechanism, which may include the pestle coupledto the motor, in the grinding chamber. The stirring mechanism mixes theparticles and the bacteria, creating a physical grinding action thatdisrupts the cells and releases their contents.

Diamond dust can be used for grinding because its irregular shape andstrength make for many sharp and durable surfaces.

After grinding the cells, any sequence of desired solutions can beflowed over the samples. In the case of RNA extraction fromcyanobacteria, a series of wash solutions is flowed through the chamberto first encourage binding of the RNA to the silica capture material andthen to clear out non-RNA impurities (e.g., proteins, lipids).

Embodiments of the present disclosure exhibit higher filtrationcapacity.

According to various aspects of the present disclosure, thethree-dimensional filter substantially increases the particles trappedat rates faster than other existing filter types, more efficiently andeffectively isolating cells.

Embodiments further exhibit higher lysis efficacy, trapping cellsbetween the abrasives, which also lyse them, therefore substantiallypreventing sample loss and contamination. Furthermore, desirable celllysis parameters can be controlled by selectively controlling the timeand rate of rotation of the pestle, and/or via abrasive particleselection and particular distribution from coarse toward opposing endsof the disruption chamber to the central, lower, intermediate, or middleregion thereof. Embodiments of the present disclosure further providefor selective collection of cells or organisms. By using different sizeparticles/abrasives we can selectively determine which size cells can becaptured and which can be passed through. Additionally, at least someembodiments are suitable for processing hard to lyse cells. For example,some cells such as plant tissue or bacteria spores are difficult to lysedue to their hard outer shell or cell walls. Abrasives can be selectedand used with an embodiment to break any cell type.

Furthermore, embodiments can be formed in an overall small disposabledevice for sample preparation.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A method for processing cells from cell solution having cells andwaste matter comprising: placing a plurality of abrasive particles in adisruption chamber; at least partially placing a pestle in thedisruption chamber; introducing cell solution including cells and wastematter into the disruption chamber following placing the abrasivestherein; selecting the abrasives to embody sizes and shapes to obstructthe cells in a space between the abrasives, and allowing therethroughthe waste matter following introduction of the cell solution to thedisruption chamber; and agitating the cells against the abrasives byactuating at least one of the disruption chamber and the pestle, theselected sizes and shapes of the abrasives configured to cut the cellsseparated from the waste matter.
 2. The method of claim 1, furthercomprising: arranging the abrasive particles in at least a first layerhaving a first density of abrasive particles and a second layer having asecond density of abrasive particles, the first density being differentfrom the second density.
 3. The method of claim 1, further comprisingcommunicating the cut cells to a binding matrix.
 4. The method of claim1 wherein the placing the plurality of abrasives includes substantiallyfilling the disruption chamber with the abrasives.
 5. The method ofclaim 1, further comprising: positioning a binding matrix between anoutlet of the disruption and an outlet port in fluid communication withthe disruption chamber.
 6. The method of claim 1, further comprising:substantially retaining the abrasives between a first mesh componenttoward a first end of the disruption chamber and a second mesh componenttoward a second end of the disruption chamber.
 7. A method ofmanufacturing an apparatus for cell capture, disruption, and extractionfrom a cell solution having cells and waste matter, the methodcomprising: forming a disruption chamber in a first fitting; at leastone of: forming an outlet port in the first fitting and coupling asecond fitting to the first fitting and forming the outlet port in thesecond fitting, the outlet port configured to be in fluid communicationwith the disruption chamber; forming an inlet port in fluidcommunication with the disruption chamber; and providing a plurality ofabrasives configured for placement in the disruption chamber prior tointroduction of the cell solution to the disruption chamber, sizing andshaping at least some of the abrasives to allow therethrough the wastematter and to obstruct the cells in a space between the abrasives thecells; providing a pestle configured to be at least partially receivedin the disruption chamber; and providing an actuation device configuredto operatively couple to at least one of the disruption chamber and thepestle to impart motion thereto, such motion agitating the abrasives, atleast some of the abrasives being comprised of a material and shape tocut the cells separated from waste matter, in response to the agitation.8. The apparatus of claim 7 wherein the providing the plurality ofabrasives includes arranging the plurality of abrasives in a pluralityof layers forming a multi-dimensional filtration and grinding matrix,the plurality of layers having at least one of distinct abrasive sizes,and shapes, at least one of the layers having the abrasives allowingtherethrough the waste matter and obstructing the cells in a spacebetween the abrasives, and at least one of the layers having theabrasives comprised of a material and shape to cut the cells.
 9. Theapparatus of claim 7, further comprising: positioning at least oneretaining member adjacent the disruption chamber.
 10. A method forextracting cell matter from a cell suspension having cell matter andnon-cell matter, the method comprising: at least partially positioning apestle in a cavity; loading a plurality of abrasives in the cavityhaving an inlet and an outlet; introducing the cell suspension to thecavity following the loading of the plurality of abrasives; selectingthe abrasives respectively having sizes and shapes such that at leastsome of the abrasives obstruct the cell matter in a space between theabrasives, filtering the cell matter from the cell suspension andallowing therethrough non-cell matter following introduction of the cellsuspension to the cavity; and imparting motion to at least one of thepestle and the cavity to agitate the abrasives, the sizes and shapes ofat least some of the abrasives configured to cut the cells separatedfrom the waste matter.
 11. The method of claim 10 wherein the loadingthe plurality of abrasives in the cavity includes arranging theplurality of abrasives in at least a first layer and a second layer,selecting the abrasives of the first layer to be larger than theabrasives in the second layer.
 12. The method of claim 11 wherein theloading the plurality of abrasives in the cavity includes arranging theplurality of abrasives in a plurality of layers including first, second,and third layers, selecting the abrasives of the second layer to besmaller than the abrasives of the first and third layer, and arrangingthe second layer between the first and third layers when the pluralityof abrasives are loaded in the cavity.